Reduction of GPSM3 expression akin to the arthritis-protective SNP rs204989 differentially affects migration in a neutrophil model.

G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.

Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance.

G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs; rs204989 and rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA)-this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378) and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1.

A new family of regulators of G-protein-coupled receptors?

Organisms as diverse as fungi and humans use G-protein-coupled receptors to control signal transduction pathways responsive to various hormones, neuroregulatory molecules and other sensory stimuli. Continual stimulation of these receptors often leads to their desensitization, which is mediated in part by the consecutive actions of two families of proteins--the G-protein-coupled receptor kinases, which phosphorylate the agonist-occupied receptors, and the arrestin proteins, which subsequently bind to the receptors. We now present evidence that a group of proteins--the G0S8/Sst2p family--may be a third class of receptor-desensitizing factors.

The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.

Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Identification of a human LIM-Hox gene, hLH-2, aberrantly expressed in chronic myelogenous leukaemia and located on 9q33-34.1.

We describe the isolation of human LH-2, a putative transcription factor containing two cysteine-rich regions (LIM domains) and a homeobox (Hox) DNA-binding domain. High levels of hLH-2 expression were observed in all cases of chronic myelogenous leukaemia (CML) tested, regardless of disease status. hLH-2 was mapped to chromosome 9Q33-34.1, in the same region as the reciprocal translocation that creates the BCR-ABL chimera of the Philadelphia chromosome (Ph'), the hallmark of CML; hLH-2 was retained on the derivative 9 chromosome and is therefore centromeric of c-ABL. The proximity of hLH-2 to the breakpoint on chromosome 9 raises the possibility of cis-activation by the t(9;22)(q34;q11) translocation. In addition to finding hLH-2 expression in all cases of CML, expression was observed in lymphoid malignancies and myeloid cell lines, but not in primary cases of acute myelogenous leukaemia. The role of hLH-2 in the development or progression of leukaemia is not known. However, hLH-2 may prove useful as a marker of CML for monitoring residual disease.

Selective regulation of N-type Ca channels by different combinations of G-protein beta/gamma subunits and RGS proteins.

We examined the effects of G-protein beta and gamma subunit heterodimers on human alpha(1B) (N-type) Ca channels expressed in HEK293 cells. All of the known beta subunits (beta1-beta5) produced voltage-dependent inhibition of alpha(1B) Ca channels, depending on the gamma subunit found in the heterodimer. beta1-beta4 subunits inhibited Ca channels when paired with gamma1-gamma3. However, beta5 subunits only produced inhibition when paired with gamma2. In contrast, heterodimers between beta5 subunits and RGS (regulators of G-protein signaling) proteins containing GGL domains did not produce inhibition of Ca channels. However, GGL domain-containing RGS proteins (e.g., RGS6 and RGS11) did block the ability of Gbeta5/gamma2 heterodimers to inhibit Ca channels. Because all of the G-protein beta subunits are found in the nervous system, we conclude that they may all potentially participate in Ca channel inhibition. The interaction of GGL-containing RGS proteins with Gbeta5gamma2 suggests a novel way in which Ca channels can be regulated.

Cooperative interaction between the DNA-binding domains of PU.1 and IRF4.

The two lymphoid-specific transcription factors PU.1 and IRF4 form a cooperative ternary complex at immunoglobulin enhancer elements such as the lambdaB and kappaE3' sites. We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding domains of the two proteins. The minimal DNA binding-domain of IRF4 was mapped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs). This domain can bind weakly to a synthetic murine lambdaB element, while IRF4 constructs that contain residues 1 to 19 require the presence of PU.1 for DNA-binding at similar concentrations. Fluorescence polarization of fluorescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs from two- to fivefold. However, all constructs bound better to the lambdaB element in the presence of the DNA-binding domain of PU.1. This cooperative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.1 and IRF4. These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves the DNA-binding domains of the two proteins. NMR spectroscopy of 15N-labelled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated proteins.

Differential expression of a basic helix-loop-helix phosphoprotein gene, G0S8, in acute leukemia and localization to human chromosome 1q31.

A basic helix-loop-helix phosphoprotein gene, G0S8, was recently isolated by differential screening of cDNA from human blood mononuclear cells stimulated with a T cell mitogen and cycloheximide. In this study, G0S8 expression was examined in normal and malignant hematopoietic cells by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). G0S8 expression was observed in most fresh samples of acute myelogenous leukemia (AML) (28/30) and most cases of adult acute lymphoblastic leukemia (ALL) (9/11) regardless of clinical classification. G0S8 mRNA was also detected in all cases tested of chronic myelogenous leukemia (CML) in blast crisis. However, G0S8 expression was not detected in CML patients in chronic phase, nor in normal bone marrow or other hematopoietic cells. G0S8 has been mapped using fluorescence in situ hybridization (FISH) to human chromosome 1q31, the same site reported for the B cell homolog BL34/1R20 and within a region implicated in the development of hematological malignancies. The consistent observation of G0S8 mRNA in patient samples of acute leukemia suggests that G0S8 expression may either play a role in leukemogenesis or represent a common consequence of dysregulated growth.

Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene.

Regulators of G-protein signaling (RGS) constitute a family of GTPase-activating proteins with varying tissue-specific expression patterns and G-protein alpha subunit specificities. Here, we describe the molecular cloning of the human RGS-r/RGS16 cDNA, encoding a predicted polypeptide of 23kDa that shows 86% identity to mouse RGS-r. Northern blot analysis shows that, like the mouse Rgs-r message, hRGS-r mRNA is abundantly expressed in retina, with lower levels of expression in most other tissues examined. Characterization of the genomic organization of the hRGS-r gene shows that it consists of five exons and four introns. We have also mapped the human RGS-r /RGS16 gene to chromosome 1q25-1q31 by fluorescence in situ hybridzation. Analysis of human ESTs reveals that at least five members of the RGS gene family map to chromosome 1q, suggesting that at least part of the RGS family arose through gene duplication. The chromosomal location, retinal abundance, and presumed function of the human RGS-r protein in desensitizing photoreceptor signaling make the RGS-r/RGS16 locus a candidate for mutations responsible for retinitis pigmentosa with para-arteriolar preservation of retinal pigment epithelium (RP-PPRE or RP12), an autosomal recessive disorder previously mapped to 1q31.

Ggamma-like (GGL) domains: new frontiers in G-protein signaling and beta-propeller scaffolding.

The standard model of signal transduction from G-protein-coupled receptors (GPCRs) involves guanine nucleotide cycling by a heterotrimeric G-protein assembly composed of Galpha, Gbeta, and Ggamma subunits. The WD-repeat beta-propeller protein Gbeta and the alpha-helical, isoprenylated polypeptide Ggamma are considered obligate dimerization partners; moreover, conventional Gbetagamma heterodimers are considered essential to the functional coupling of Galpha subunits to receptors. However, our recent discovery of a Gbeta5 binding site (the Ggamma-like or "GGL" domain) within several regulators of G-protein signaling (RGS) proteins revealed the potential for functional GPCR/Galpha coupling in the absence of a conventional Ggamma subunit. In addition, we posit that the interaction between Gbeta5 isoforms and the GGL domains of RGS proteins represents a general mode of binding between beta-propeller proteins and their partners, extending beyond the realm of G-protein-linked signal transduction.

A set of human putative lymphocyte G0/G1 switch genes includes genes homologous to rodent cytokine and zinc finger protein-encoding genes.

Lymphocyte G0/G1 switch genes (G0S genes) are potential oncogenes and may regulate, be regulated by, or be coordinately regulated with, latent lymphotropic viruses. To identify these genes, a cDNA library was prepared from blood mononuclear cells that had been cultured for 2 hr with a T-cell mitogen (lectin) and cycloheximide. Eight differentially hybridizing recombinants were characterized by RNA and DNA blotting and sequencing. One cDNA (G0S7) corresponded to the oncogene c-fos. Another cDNA (G0S19) was homologous (70%) to a cDNA encoding a murine inhibitor of stem cell proliferation (the cytokine MIP1 alpha) and, less closely, to other members of the "small inducible" secreted protein-encoding gene family. Whereas cDNA hybridization to genomic DNA blots indicated a small subfamily of G0S19 genes, simple patterns of bands indicated that most cDNAs, including G0S30 cDNA, corresponded to single-copy genes. The 3' noncoding sequence of G0S30 cDNA was homologous (87-89%) to the 3' noncoding sequences of certain rodent genes (NGFI-A, Krox24, EGR1) that encode zinc finger proteins (putative transcriptional regulators). This degree of evolutionary conservation suggests an important function for the 3' noncoding region. The 3' noncoding regions of some cDNAs contained the TTATTTAT (mRNA destabilization) element. The corresponding RNAs each formed doublets in agarose gels. Previous studies of c-fos RNA from HeLa cells indicate that this is due to cycloheximide-dependent stabilization of poly(A) tails. Our results reveal the power of cycloheximide enrichment in isolating what would appear to be significant low-abundance mRNAs.

A human gene encoding a putative basic helix-loop-helix phosphoprotein whose mRNA increases rapidly in cycloheximide-treated blood mononuclear cells.

G0S8 is a member of a set of putative G0/G1 switch regulatory genes (G0S genes) selected by screening cDNA libraries prepared from blood mononuclear cells cultured for 2 hr with lectin and cycloheximide. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 211 amino acids, distributed across 5 exons. The 24-kD protein has a basic domain preceding a potential helix-loop-helix domain which contains a QTK motif found about 60 amino acids from the carboxyl terminus in the loop region of several helix-loop-helix proteins. There are potential phosphorylation sites for protein kinase C, creatine kinase II, and protein tyrosine kinases and regions of sequence similarity to helix-loop-helix proteins, tyrosine phosphatases, and RNA and DNA polymerases. The genomic sequence contains a CpG island, suggesting expression in the germ line. Potential binding sites for transcription factors are present in the 5' flank and introns; these include Zif268/NGFI-A/EGR1/G0S30, NGFI-B, Ap1, and factors that react with retroviral long terminal repeats (LTRs). There are several potential interferon response elements and a serum response element in the 3' flank overlapping a region of similarity to a cytomegalovirus immediate-early gene enhancer. Many of these motifs are found in immediate-early G0/G1 switch genes; however, we were unable to demonstrate an increase in G0S8 mRNA in response to lectin alone. Sequence similarities are noted between G0S8 and a variety of genes involved in the immune system, in the regulation of retroviruses, and in the cell cycle.

RAMHA: a PC-based Monte-Carlo simulation of random saturation mutagenesis.

Random mutagenesis is a powerful tool in protein structure-function analyses. One approach to random mutagenesis is the de novo synthesis of polypeptide-encoding oligodeoxy-nucleotides using doped nucleoside phosphoramidites. A Turbo PASCAL program, RAMHA, is described for modeling such mutagenesis. Upon entering the target sequence and the desired level of nucleotide contamination, RAMHA performs a Monte Carlo simulation of the mutagenesis, compiling statistics on the similarity of resultant mutant polypeptides to the wild-type sequence, the frequency of premature open-reading frame terminations, and other relevant outcomes. Simulated mutagenesis of two DNA targets has led to the development of two different strategies to avoid the random introduction of stop codons within mutagenized gene segments.

G-protein signaling: back to the future.

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Galpha.GDP/Gbetagamma heterotrimers to promote GDP release and GTP binding, resulting in liberation of Galpha from Gbetagamma. Galpha.GTP and Gbetagamma target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Galpha and heterotrimer reformation - a cycle accelerated by 'regulators of G-protein signaling' (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) beta is activated by Galpha(q) and Gbetagamma, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Galpha nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways.

Whither goest the RGS proteins?

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits - the "regulator of G protein signaling" or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA.

In Drosophila, phototransduction is mediated by G(q)-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.

Molecular cloning and expression analysis of rat Rgs12 and Rgs14.

We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologue of rRgs12 by alignment of cosmid sequences in the database which map the human RGS12 gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology to rRgs14 which map to human chromosome 5qter. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.